A novel enterovirus in lambs with poliomyelitis and brain stem encephalitis

Abstract An Austrian organic dairy sheep farm experienced cases of recumbency and sudden deaths in 3‐ to 4‐week‐old lambs. Two animals were subjected to thorough clinical and pathological investigations. Pathohistological analysis identified severe nonsuppurative myelitis and mild nonsuppurative encephalitis. A reverse‐transcription quantitative PCR (RT‐qPCR) assay for the recently discovered ovine picornavirus causing comparable lesions scored negative. By next‐generation sequencing‐based metagenomics, a nearly complete genome of a novel enterovirus could be detected and assembled. In situ hybridization using a specifically designed probe revealed robust signals in affected motoneurons of the spinal cord suggesting a causative role of the novel virus.

The authors suggested that this infection maybe widespread, but usu-This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH ally subclinical and self-limiting, particularly in older animals. The fact that the disease was only recognized in artificially reared lambs, which did not receive colostrum, indicated likely failure of colostral antibody transfer. It has been hypothesized that clinical disease due to this virus is rare because of herd immunity, which is not transferred to lambs deprived of colostral uptake. These cases had been found exclusively in the United Kingdom so far. In the present contribution, we describe an episode of ataxia progressing into paralysis and recumbency in a herd of lambs in Austria, with pathological lesions indistinguishable from the ones reported earlier.

Herd history
The cases occurred in an organic dairy sheep farm located in the northern area of Lower Austria. The Lacaune dairy sheep herd is free of  (Schoiswohl et al., 2020). The 130 dairy sheep are housed in a free stall, which was built in 2018. All ewes give birth to the lambs in late autumn. In case newborn lambs show a well-developed mother-lamb relationship (physiological general behaviour, adequate colostrum/milk intake), they are allowed to stay with their ewes for 5-10 days following birth. Otherwise, they are artificially reared which means that they are separated and fed from an artificial milk provider. Female lambs are kept as young stock at the farm and the majority of males are sold for fattening to another farm at the age of 10 days or stay at the farm to be sold with approximately 40 kg.

Case presentation
On 5 November 2019, the farmer consulted the University Clinic for Ruminants, Vienna since he recognized sudden deaths in male and female lambs aged between 3 to 4 weeks, all born between 12 October and 22 October. The sudden deaths were observed solely at the farm of origin, not at the fatteners. Four lambs died within 24 h, initially showing elevated body temperature followed by decreased body temperature and progressive neurological symptoms. The first signs were hind limb weakness, followed by paralysis of the hind limbs, with preservation of milk and feed ingestion ( Figure 1). Later, the animals became recumbent, showed torticollis and/or opisthotonus and died within 24 h. One female (lamb 1; 17 days; 8 kg) and one male (lamb 2; 24 days; 9 kg) Lacaune lamb were presented at the University Clinic for Ruminants for herd diagnosis. One of the animals was artificially reared due to postnatal weakness and questionable colostrum intake. The medical history was not clear enough to verify which of the animals had a lack of colostrum intake. Lamb 1 showed hind limb paralysis (Figure 1), no deep sensibility and muscular tone, and an increased body temperature

Necropsy and histological examination
Both animals were necropsied according to standard protocols. A set of tissue samples including brain and spinal cord were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 2-3 µm, and stained with haematoxylin and eosin.

Next-generation sequencing and sequence analyses
Frozen brain and spinal cord samples of lamb 1 were used for nextgeneration sequencing (NGS)-based metagenomics. Sample preparation was performed as described previously (Wylezich et al., 2018) with a few changes. Briefly, after disintegration of the brain tissue by cryoPREP (Covaris) treatment, RNA was extracted with an RNAdvance Tissue Kit (Beckman Coulter) according to the manufacturer's instructions on a KingFisher Flex (Thermo Fisher Scientific) instrument. The extracted RNA was quantified with a Nanodrop ND1000 (PeqLab), and 500 ng RNA was used as input for cDNA and second strand synthesis using a SuperScript IV First-Strand Synthesis System (Life Technologies) and the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB). Thereafter, the resulting doublestranded cDNA was fragmented with a Covaris M220 focused sonicator (Covaris), and the fragmented DNA was used as input for library preparation, which was performed with a GeneRead DNA Library L Core Kit (Qiagen) and an Ion Xpress Barcode Adapter (Life Technologies). The generated Ion Torrent compatible libraries were quantified with the KAPA Library Quantification Kit -Ion Torrent Universal (Roche) and then sequenced with an Ion Torrent S5 XL (Thermo Fisher Scientific) on an Ion 530 chip in 400 Bp mode according to the manufacturer's instructions. For the initial taxonomic binning, the resulting dataset was analyzed using RIEMS (Scheuch et al., 2015). Since the virus content of the library was low, additional sequencing (Ion Torrent S5 XL, Ion 540 Chips) was performed as per the manufacturer's instructions in order to generate enough data for the assembly of a complete viral genome. The genome sequence was assembled from the combined datasets applying an iterative hybrid approach. First, reads representing the enteroviral genome were identified using Diamond blastx (Buchfink et al., 2015) and all to date available related picornaviral sequences. These reads were assembled de novo using Newbler (v 3.0; Roche). In subsequent mappings (Newbler, v3.0; Roche), additional reads were identified and the genome sequence was reassembled until the nearly complete genome sequence was obtained.
The sequence is available from the INSDC databases under study accession PRJEB47260.

Phylogenetic analysis
For

Reverse-transcription quantitative PCR
This method was used for investigating the tissue distribution of the virus in formalin-fixed and paraffin wax-embedded tissue samples.
From each block, five sections with a thickness of 10 µm were placed in a 1.5 ml tube. RNA was extracted from FFPE material using the truXTRAC FFPE total NA Kit (Covaris) in combination with the Agencourt RNAdvance Tissue Kit (Beckman Coulter) and a KingFisher Flex instrument (Thermo Fisher Scientific). From the initial draft sequence, a reverse-transcription quantitative PCR (RT-qPCR) assay was designed.
As a positive control, a gBlock oligo (Integrated DNA Technologies) was used. As RT-qPCR was performed with a SensiFAST Probe No-ROX Kit (Meridian) according to the manufacturer's instructions. The PCR was run as a duplex assay containing an internal control assay as described (Hoffmann et al., 2006) and for the detection of the novel enterovirus followed by 20 s at 60 • C (elongation; data acquisition).

RESULTS AND DISCUSSION
Necropsy of both animals did not reveal any obvious abnormalities.
The histological lesions were identical in both animals and were confined to the central nervous system. The spinal cord was most severely affected. All levels showed a severe nonsuppurative myelitis, which was characterized by excessive necrosis (Figure 2a)  The lesions were particularly severe in the grey matter, but the perivascular cuffs extended into the white matter as well. The lesions in the brain were less pronounced and restricted to brainstem areas, such as thalamus, midbrain, medulla oblongata, and cerebellar roof nuclei.
Here, perivascular cuffs and multifocal gliosis were the most prominent findings.
Due to the striking histological similarities to a recently described picornavirus infection, native brain and spinal cord samples of lamb 1 were subjected to a specific RT-qPCR assay (Forth et al., 2019), but the result was clearly negative. As the lesions were highly suggestive of a It comprises one open reading frame that encodes one polyprotein with 2172 amino acids. Phylogenetic analysis revealed enteroviruses derived from goat and sheep as the closest relatives (Figure 3). These By RT-qPCR, viral RNA was found in all examined spinal cord samples and in caudal brain areas, which corresponds well to the ISH results. In the majority of extraneural tissues, no viral RNA was identified; a low amount of viral RNA was detected in a liver, muscle, and intestinal sample of one animal, each (Table 1). In both animals, the concentration of viral nucleic acids in neural tissues was higher with a Cq difference of at least 5 between the highest load in neural compared to extraneural tissue.
In the last decade, particularly NGS-based metagenomics contributed to significant progress in the elucidation of previously elusive causes of nonsuppurative encephalomyelitides in many animal species.
Especially enteroviruses are known to cause encephalomyelitis in several vertebrate species, and it is intriguing that there is a particular tropism to the spinal cord motor neurons. Brain lesions are comparatively less common. Two prominent disease entities caused by this group of viruses are poliomyelitis of humans (Melnick, 1996) and Teschen disease of pigs (Cantile & Sameh, 2016). The histological lesions of these entities are characterized by neuronal necrosis, neuronophagia, and glial nodules predominantly in the ventral horns of the spinal cord (Melnick, 1996;Pogranichniy et al., 2003). This seems to be a consistent feature of neurotropic enterovirus infections, 3) have been declared eradicated due to successful vaccination campaigns (Chard et al., 2020;Nathanson & Kew, 2010). In pigs, historically, porcine enteroviruses (serotypes 1 to 11) have been members of the genus Enterovirus in the Picornaviridae family. Recent reclassification has resulted in the establishment of two new genera, the Teschovirus genus (serotypes 1-7 and 11-13, now porcine teschovirus) and the Sapelovirus genus (serotype 8, now porcine sapelovirus A). Porcine enterovirus serotypes 9 and 10 (PEV-9 and PEV-10) remain in the Enterovirus genus (Alexandersen et al., 2019).
In accordance with the variation of human and porcine enteroviruses, it is very likely that a larger number of ovine enterovirus exist which are generally able to induce myelitis in lambs, probably with geographic restrictions. Within a short time span, two of them have been discovered, but it can be expected that more will emerge.
Novel enteroviruses may also be generated due to intraspecies and even interspecies recombination events. Boros et al. (2012) presented Also, the circumstances of enterovirus pathogenicity (across species) are very similar. The viruses are widespread within their host species and the resulting herd immunity prevents the viruses from spreading from the intestine to the nervous system. This is the reason why enterovirus-associated cases of (encephalo)myelitis are quite rare. Nervous system manifestation occurs in individuals with immunity gaps, either due to failure of colostrum uptake or at the time of weaning, when maternal immunity wanes and active immunity is not yet fully developed. Also, in the cases described here, a lack of colostrum uptake was evident in at least one lamb. Still, it cannot be ruled out that further lambs received insufficient colostrum since the lambs usually stay with their mothers for 5-10 days, and feed uptake is solely visually monitored and not further verified.
Taken together, this report describes a novel enterovirus which may be common in sheep and seems to be able to cause myelitis with severe motoneuron damage in colostrum-deprived lambs. Thus, sheep farmers should be instructed that colostrum uptake has to be secured in order to provide adequate immunity to these potentially deleterious viral agents.

ACKNOWLDGEMENTS
We thank Klaus Bittermann for his help with the image layout. We are grateful to Patrick Zitzow for excellent technical support.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ETHICS STATEMENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. The authors confirm that no ethical approval was required as this work was carried out according to the rules pertaining to the clinical and diagnostic services of Vetmeduni Vienna.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.